This invention relates to novel substituted heterobicyclic pyrimidine compounds, in particular substituted pyrazolopyrimidine oxindoles, that act as inhibitors of glycogen synthase kinase 3 and cyclin dependant kinase 5.
Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase composed of two isoforms (α and β) encoded by different genes. GSK3 is highly expressed in the central and peripheral nervous system, with GSK3β predominating in the brain. Both isoforms of GSK3 phosphorylate and regulate the activity of several protein substrates, including glycogen synthase, β-catenin, pyruvate dehydrogenase, elongation intiation factor 2b, and tau. GSK3 is regulated by insulin, which stimulates glycogen synthesis via receptor activation of PI3 kinase and protein kinase B. PKB phosphorylates GSK3β on serine 9, resulting in its inactivation. Insulin also activates protein phosphatase 1. Both of these actions of insulin lead to dephosphorylation and activation of glycogen synthase (Srivastava and Pandey, Mol Cell Biochem. 182:135-141, 1998; Cohen, Biochem Soc Trans., 21:555-567, 1993), and production of glycogen from glucose. β-catenin degradation is increased following phosphorylation by GSK3 (Ikeda, et al., EMBO, 17:1371-1384, 1998). The reduction in available β-catenin may increase the sensitivity of neurons to amyloid β (Aβ) toxicity (Zhang, et al., Nature, 395:698-702, 1998). GSK3β also phosphorylates pyruvate dehydrogenase and prevents the conversion of pyruvate to acetyl CoA (Hoshi, et al., PNAS, 93:2719-2723, 1996). This acetyl CoA is critical for the synthesis of acetylcholine, the loss of which is implicated in the cognitive decline in Alzheimer's Disease (AD). GSK3α regulates production of Aβ from the amyloid precursor protein (Phiel, et al., Nature. 423:435-9, 2003). Two proteases, β- and γ-secreatase liberate the amino and carboxy terminus (respectively) of Aβ. In a concentration dependant manner, Aβ precipitates into toxic, fibrillary species in the AD brain and is thought to lead to additional sequalae of the disease. Phosphorylation of eIF2B by GSK-3β reduces protein translation. eIF2B activation by IGF1 is mediated by the inactivation of GSK3β (Welsh, et al., FEBS Letts, 421:125-130, 1997). The role of tau phosphorylation by GSK3 will be discussed following description of CDK5.
Cyclin Dependant Kinase 5 (CDK5) is also a serine/threonine protein kinase, and is structurally related to GSK3. CDK5 activation predominates in the nervous system due to expression of p35, an accessory protein related to cyclins and necessary for CDK5 activity (Dhavan and Tsai, Nat Rev Mol Cell Biol, 2:749-759, 2001). Unlike CDK1, 2, 4, and 6 which are active in the cell cycle, CDK5 is activated in neurons after cell division has ended, following differentiation and expression of p35. CDK5 activity is regulated by expression of p35 and a calpain-cleaved form of p35, known as p25 (Patzke and Tsai, J Biol Chem, 277:8054-8060, 2002). The generation of p25 leads to increased and mislocalized CDK5 activity since 1) p25 is missing the membrane localizing portion found in p35, and 2) p25 has a longer resident half life in the cytoplasm. CDK5 phosphorylates a number of substrates including DARPP-32, NR2a (NMDA receptor subunit), MEF-2, PSD-95, synaptojanin-1, CRMP2, and tau. DARPP-32 phosphorylation by CDK5 at thr75 leads to the inhibition of PKA in the dopamine 1 receptor (D1) signaling cascade, thereby inhibiting D1 signaling (Bibb, et al., Nature, 402:669-671 1997). Facilitation of D1 signaling may be useful for the treatment of depression or Parkinson's Disease (Chergui, et al., PNAS, 10:2191-2196, 2004). NR2a phosphorylation by CDK5 modulates long term potentiation and may induce apoptotic cell death following ischemia (Wang, et al., Nat Neurosci., 6:1039-47, 2003). CDK5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses, providing a possible mechanism for rapid changes in density and/or number of synaptic receptors (Morabito, et. al., J Neurosci., 24:865-876, 2004). CDK5 also phosphorylates the presynaptic phosphatase synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes (Lee, et. al., PNAS, 101:546-551, 2004). CRMP2 is also phosphorylated by CDK5, leading to a reduction in CRMP-tubulin binding affinity and modulating growth cone collapse. CDK5 primarily phosphorylates CRMP2 at Ser522 and GSK3β secondarily phosphorylates at Thr509. Dual-phosphorylated CRMP2 is recognized with the antibody 3F4, highly reactive with the neurofibrillary tangles (NFT) of AD brain (Uchida, et al., Genes Cells, 10:165-179, 2005). Overall, the role of CDK5 in synaptic formation and function is well substantiated.
Experimental evidence supports a role for both GSK3 and CDK5 in the tangle and plaque pathology of AD, namely in the tau hyperphosphorylation that leads to NFT formation. AD brain is characterized by intracellular NFTs and extracellular senile plaques consisting of Aβ deposits. Both of these protein aggregates are thought to precipitate the neuronal and synaptic loss, leading to the memory loss and cognitive decline of AD (Hardy, J Mol Neurosci, 20:203-6, 2003).
NFTs are composed of hyperphosphorylated, aggregated forms of the neuron specific, cytoskeletal protein tau (Cairns, et. al., J Pathol, 204:438-449, 2004). The primary function of tau is to stabilize neuronal microtubules, to maintain axonal architecture, and to allow transport of materials both from the cell body to the synapse, and from the synapse back to the cell body. In AD, tau is hyperphosphorylated at many serine/threonine residues, leading to poor binding of tau to the microtubule and loss of trophic interplay between the cell body and the synapse. NFTs represent one of the characteristic features of the AD brain, and are also present in the brains of individuals with progressive supranuclear palsy, frontotemporal dementia with parkinsonism-17, Neimann-Pick's disease, corticobasal degeneration, amyotrophic lateral sclerosis, dementia puglistica, etc.
NFTs are composed of insoluble aggretates of tau protein, hyperphosphorylated on many serine and threonine residues and formed into paired helical filaments. The hyperphosphorylation of tau results in a lower affinity for the microtubule and may represent the first step toward aggregate formation. Both CDK5 and GSK3 phosphorylate tau in both cell-free and cell-based in vitro systems at many of the same sites present in the AD brain. Antibodies directed against both GSK3 (Pei, et. al., J. Neuropath. Exp. Neurol., 58: 1010-1019, 1999) and CDK5 (Pei, et. al., Brain Res, 797:267-277, 1998) decorate the NFTs in the AD brain, demonstrating the close association between these kinases and the hyperphosphorylated tau that comprises the tangles. Overexpression of either kinase activity in transgenic animal models (Lucas, et al., EMBO J., 20:27-39, 2001; Cruz, et al., Neuron, 40:471-83, 2003) also demonstrates their ability to hyperphosphorylate tau (both CDK5 and GSK3) and cause the formation of mature NFTs and neuronal loss (CDK5). Phosphorylation by GSK3 at many epitopes requires prior phosphorylation by a so called “priming” kinase C-terminal to the GSK3 phosphorylation site (Cohen and Goedert, Nat Rev Drug Discov, 3:479-487, 2004). Interestingly, CDK5 has been implicated as a priming kinase (at phosphoserine 235) for GSK3, which acts to phosphorylate threonine 231, a site that is phosphorylated early in the progression of AD NFT pathology (Augustinack, et. al., Acta Neuropathol (Berl). 103:26-35, 2002; Li T, Hawkes C, Qureshi H Y, Kar S, Paudel H K, Biochemistry, 45:3145-4154, 2006).
Other disease states in which GSK3 is thought to play a role include cerebral ischemia. GSK3 activity is increased in cellular and animal models of both neurodegeneration and apoptosis, such as cerebral ischemia (Bhat, et al., PNAS, 97:11074-11079, 2000). Lithium, as a representative GSK3 inhibitor, is neuroprotective in these models (Ren, et al., PNAS, U S A, 100:6210-6215, 2003). Lithium inhibits GSK3 at concentrations also known to be therapeutic in bipolar disorder (Gould, et al., J Clin Psychiatry, 65:10-21, 2004), implicating GSK3 inhibition as a therapeutic avenue in this disease.
GSK3 activity is increased in peripheral lymphocytes and brains of patients with schizophrenia, as evidenced by reduced levels of both the upstream inhibiting kinase AKT1, and the inhibitory ser9 phosphorylation of GSK3β (Emamian, et al., Nat Genet, 36:131-137, 2004) Clinical treatment leads to normalization of this pathway.
Diabetes mellitus type 2 is characterized by reduced insulin production due to loss of pancreatic beta cells following a period of reduced insulin sensitivity. With the insulin receptor signaling dysfunction that is also present in the disease, direct inhibition of GSK3 has been hypothesized to relieve the hyperglycemia and allow for normal glycogen synthesis and glucose utilization (Wagman, et al., Curr Pharm Des., 10:1105-1137, 2004).
These compounds as GSK3 inhibitors are indicated to be useful for the treatment and/or prophylaxis of conditions in which there is a need for inhibition of GSK3, such as diabetes, conditions associated with diabetes, chronic neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, progressive supranuclear palsy, subacute panencephalitic parkinsonism, postencephalitic parkinsonism, dementia puglistica, guan-parkinsonial dementia complex, Pick's disease, corticobasal degeneration, frontotemporal dementia with parkinsonism, Huntington's disease, AIDS associated dementia, amyotrophic lateral sclerosis, multiple sclerosis, and neurotraumatic diseases such as acute stroke, mood disorders such as schizophrenia and bipolar disorders, promotion of functional recovery post stroke, cerebral bleeding (solitary cerebral amyloid anigopathy), hair loss, obesity, atherosclerotic cardiovascular disease, hypertension, polycystic ovary syndrome, syndrome X, ischemia, traumatic brain injury, cancer, leukopenia, Down's syndrome, Lewy body disease, inflammation and immunodeficiency.
These compounds as CDK5 inhibitors are indicated to be useful for the treatment and/or prophylaxis of conditions in which there is a need for inhibition of CDK5 such as the chronic neurodegenerative diseases Alzheimer's disease, Parkinson's Disease, progressive supranuclear palsy, subacute panencephalitic parkinsonism, postencephalitic parkinsonism, dementia puglistica, guan-parkinsonial dementia complex, Pick's disease, corticobasal degeneration, frontotemporal dementia with parkinsonism, Huntington's disease, AIDS-associated dementia, amyotrophic lateral sclerosis and mood disorders such as depression.
Thus, there is a need for novel classes of compounds that possess the beneficial properties. It has been discovered that a class of compounds, referred to herein as substituted heterobicyclic pyrimidine compounds, in particular substituted pyrazolopyrimidine oxindoles, are useful as agents for treating or preventing various diseases or disorders disclosed herein.